Halogen atoms are important structural features found in a variety of natural and synthetic compounds of medicinal interest. Among enzymes used by biological systems to install halide functionality into natural products, the non-heme iron (II) halogenase family is of particular interest. These enzymes catalyze the chlorination of unactivated C-H bonds using a high valent iron (IV) oxo intermediate, a strategy shared by a related enzyme class, the non-heme iron (II) hydroxylases. The similar reactivity of these enzymes, as well as their structural homology, has led to the hypothesis that the halogenase family evolved from hydroxylases. As a means of probing the validity of this hypothesis, one can envision engineering a non-heme iron (II) hydroxylase into a halogenase. Two approaches for the engineering of the well-characterized asparagines hydroxylase AsnO are proposed: minimalist active site redesign and directed evolution. Minimalist active site redesign will employ structural, mechanistic and computational tools to identify selected target residues within AsnO for mutation. The directed evolution approach utilizes various mutagenesis techniques, including random and saturation mutagenesis, to generate a library of AsnO mutants, which will be screened for halogenase activity using one of two proposed spectrophotometric methods. The most promising variants will be subjected to further rounds of mutation and screening to provide an artificial halogenase suitable for structural and spectroscopic studies. PUBLIC HEALTH RELEVANCE Many of the halogenated metabolites found in nature possess intriguing biological activity which makes them interesting targets for the discovery and development of new medicines. In addition to naturally occurring compounds, many important man-made pharmaceuticals contain halides. In both cases, these atoms often have a dramatic, positive influence on the physiochemical properties of the molecule. Improvements in potency, oral bioavailability, cell-permeability, and metabolic stability may all result from the addition of a halide atom into a drug scaffold. As a result the development and mechanistic investigation of methods, both chemical and biological, for the selective installation of chlorine and other halogen atoms into molecules is an important area of research.